The present invention relates to peptides, referred to as CBD-1, CBD-2, CBM-1/TH-1, CBM-1/TH-2, CBM-2/TH-1, CBM-2/TH-2 and C-20 peptides, which are immunogenic and elicit a protective immune response against HIV infection in vitro. Pharmaceutical or therapeutical compositions and vaccines comprising these antigenic peptides are also encompassed by the present invention, as well as neutralizing antibodies which inhibit HIV infection and, when added to already infected cells, cause the production of defective HIV particles. Methods for diagnosis of HIV are also disclosed.
The gp41 molecule of HIV-1 is a transmembrane protein with several important features within its ectodomain (amino acids residues 512 to 681; the numbering is according to Dong et al. (1). First, the amino terminus of gp41, created by proteolytic cleavage of the gp160 precursor, contains a hydrophobic, glycine-rich “fusion” peptide that is essential for membrane fusion. Second, there are two α-helix containing domains at the N- and C-terminal of gp41 with a sequence motif characteristic of coiled coils. Between these two α-helix domains there is the immunodominant domain including a small loop. The two α-helix containing domains are arranged in very stable six-helix bundles. Three N-helices (amino acids 545-590) form an interior, parallel coiled-coil trimer, while three C-helices (amino acids 628-661) pack in an oblique, antiparallel manner into highly conserved, hydrophobic grooves on the surface of this trimer. This structure likely represents the core of fusion-active gp41 (2, 3). A synthetic peptide referred to as T-20 (amino acids 638-673 of gp41), corresponding to the C-helix domain of gp41, was shown to block HIV-induced fusion entry in cell cultures and HIV-infection in virus-infected individuals (4, 5). The mechanism of this inhibition is due to the binding of the T-20 peptide to the N-helix domain. Thus, by blocking the entry of HIV into normal cells is one means to arrive at inhibiting HIV-infection.
Lipid rafts are known to play an important role during the HIV entry process into target cells (6). Lipid rafts are glycolipid-enriched membrane microdomains, which are fundamental in the lateral organization of the plasma membrane by forming platforms that are implicated in the clustering of membrane proteins, endocytosis, signal transduction and membrane trafficking (7, 8, 9, 10). Lipid rafts contain sphingolipid and cholesterol-based structures that are associated with specific membrane proteins such as glycosylphosphatidylinositol (GPI)-linked proteins CD59 and CD90 Lipid rafts containing caveolin as the defining protein component are known as caveolae. Caveolins bind cholesterol directly, which stabilizes the formation of caveolin homo-oligomeric complexes (10). While lipid rafts appear to be small in size and dispersed all around the plasma membrane of non-polarized cells, the interaction of raft-associated proteins with their ligands or their cross-linking with antibodies lead to the oligomerization of raft components (9, 11).
Lipid rafts with their associated proteins can be isolated by virtue of their insolubility in the non-ionic detergent, Triton X-100® (8). A number of pathogens including viruses have been reported to use lipid rafts and caveolae as the endocytosis route or by exerting their pathogenic effects (7, 12). In the case of HIV-1, several groups have reported the implication of lipid rafts in the viral entry (6, 13, 14) and budding processes (15,16,17).
HIV-1 infects target cells by the capacity of its envelope glycoproteins gp120-gp41 complex to attach cells and induce the fusion of virus to cell membranes, a process which leads to virus entry (18). The external envelope glycoprotein contains the binding site for the CD4 receptor and an hypervariable region of about 36 amino acids referred to as the V3 loop. The transmembrane glycoprotein contains a potential fusion peptide at its amino terminus, which is implicated in the membrane fusion process. The external and transmembrane glycoproteins (gp120-gp41 for HIV-1) are associated in a noncovalent manner to generate a functional complex. gp120 determines viral tropism by binding to target-cell receptors, while gp41 mediates fusion between viral and cellular membranes. The receptor complex essential for HIV entry into cells consists of the CD4 molecule and at least one member of the chemokine receptor family; CCR5 is the major coreceptor for macrophage-tropic HIV-1 isolates (R5), whereas for T-lymphocyte-tropic isolates (X4) the major coreceptor is CXCR4 (19, 20). Several observations have also pointed out that the initial attachment of HIV particles to target cells occurs through the co-ordinated interaction of the V3 loop with the cell-surface-expressed heparan sulfate proteoglycans and nucleolin (21, 22, 23, 24).
Consistent with the implication of lipid rafts in the HIV-1 entry process, CD4, CXCR4 and CCR5 partition and signal in rafts after gp120-induced clustering (14) or cross-linking of the cell-surface-bound HIV particles (6), while the functioning of CD4 and chemokine receptors appears to require their association in lipid rafts (25, 26). Depletion of cellular cholesterol by the drug, β-cyclodextrin, renders primary cells and cell lines highly resistant to HIV-1 mediated syncytium formation and also to infection by both X4 and R5 HIV-1 strains (27). Epithelial transcytosis of HIV-1 also uses the lipid raft pathway as a transport mechanism to get from the apical side to the basolateral side of the cell (28). In addition, lipid rafts have been shown to play a critical role in HIV-1 assembly and release that takes place at the plasma membrane. Both HIV-1 Gag and envelope protein appear to be associated with lipid rafts, a process which is necessary for the assembly of HIV proteins at the plasma membrane and for the budding of virions (16, 17). Recently, the coexpression of caveolin-1 with HIV-1 has been reported to block virus production mostly by inhibiting viral protein synthesis, although some minor effects on HIV budding were not excluded (15). The region in caveolin-1 responsible for this latter inhibitory effect was found to be the hydrophobic, membrane-associated domain (residues 101 to 135), whereas the first 100 N-terminal amino acids, which include the oligomerization and scaffolding domains, were shown to be dispensable (15). Finally, transcytosis of HIV across epithelial cells have been shown to be mediated by the capacity of virus particles to bind glycosphingolipid galactosyl ceramide receptors in caveolae (28).
Caveolae are a specialized form of lipid rafts defined by the presence of a specific protein marker, caveolin. They have a unique lipid composition, mainly composed of cholesterol and sphingolipids (10). There are multiple forms of caveolin: caveolin-1 and caveolin-2 are expressed as stable heterooligomeric complexes within most cell types, while caveolin-3 is restricted to striated muscle cells. Recent observations have pointed out that cells implicated in HIV infection, such as lymphocytes, macrophages, and dendritic cells express caveolin-1 at the cell surface (Harris et al., 2002). Caveolin-1 contains 178 amino acid residues. Its central hydrophobic domain (amino acids 102-134) is thought to form a hairpin-like structure within the membrane with both the N-terminal domain (amino acids 1-101) and the C-terminal domain (amino acids 135-178) facing the cytoplasm. A short domain at amino acids 82 to 101 in caveolin has been defined as caveolin-scaffolding domain which is responsible for the formation of multivalent homo-oligomers of caveolin and also represents a domain implicated in the interaction of caveolin with different ligands (for reviews see (10, 41)). By using caveolin-scaffolding domain as a receptor, Lisanti and collaborators have selected caveolin-binding peptide motifs from phage display libraries (40, 41). Two related caveolin-binding motifs have been defined: φXφXXXXφ and φXXXXφXXφ, where φ is an aromatic amino acid Trp, Phe, or Tyr, whereas X is any other amino acid.
In view of the implication of lipid rafts in HIV entry and budding process (12), any substance that interferes with the functioning of lipid rafts during HIV infection, would inhibit HIV entry and/or budding process and thus be an effective tool to treat and/or prevent HIV-infection.
Ever since the discovery and isolation of LAV by Barre-Sinousi, Chermann and Montagnier in 1983 at the Pasteur Institute a search for effective treatment without major side effects and prevention of AIDS has been elusive.
Treating patients with AIDS with a combination of reverse transcriptase and drugs that target HIV's protease enzyme, known in the art as “highly active antiretroviral therapy,” is effective to drive the viral load in blood to low levels.
Thus, since 1996, antiretroviral drugs such as zidovudine (AZT), ritonavir, saquinavir, lamivudine, amprenavir, abacavir, idinavir, nelfinavir and the like, were generally used in triple-drug therapy using two reverse transcriptase inhibitors and one protease inhibitor, to reduce the amount of HIV in patients. However, none of these drugs entirely eliminates the virus.
Moreover, there remains serious problems associate with the triple-drug therapy. Not only must an HIV-infected person take the drugs on a consistent schedule and for the duration of life, but these drugs are not only quite expensive ($10,000 annually or more), but toxic. Due to their toxic nature, antiretroviral drugs have known side effects which include nausea, vomiting, diarrhea, anemia, lipodystrophy, diabetes-like problems, brittle bones, numbness, tingling or pain in the hands or feet, and heart disease. As a result of these side effects many AIDS patients stop taking their medication.
Besides their toxic effects, one of the major difficulties with highly active retroviral therapy is drug resistance. Since HIV is known to constantly mutate, billions of new HIV viruses are produced in the body every day. These mutations change parts of the virus often rendering the drugs ineffective.
A better solution to treat HIV is to entirely eliminate the virus by the use of immunogens that can induce humoral and cellular immune responses against HIV (42). In view of this, epitopes in the surface gp120 and transmembrane gp41 envelope glycoproteins of HIV-1 have been investigated in great detail.
The principal targets of neutralizing antibodies against HIV-1 are the surface and transmembrane envelope glycoproteins gp120 and gp41 (43). The HIV-1 gp120 contains three major targets for the action of neutralizing antibodies, the CD4 binding domain, the third hypervariable domain referred to as the V3 loop, and a conserved region between the V1/V2 and V3 loop that appears to be responsible for the binding of R5/X4 virus to their respective chemokine receptor. Several neutralizing monoclonal antibodies have been raised against gp120, whereas against gp41 only neutralizing human monoclonal antibodies have been isolated from HIV-infected individuals. One such human monoclonal antibody is MAb CL3 that recognizes 10 amino acids within the immunodominant region in gp41 (44). Another type of neutralizing human monoclonal antibodies are directed against the membrane-proximal external region of gp41 (amino acids 657-671) containing the ELDKWA epitope of MAb 2F5, known as the “Katinger epitope” (45). However, it should be noted that synthetic peptides corresponding to these specific epitopes in gp41 fail to elicit the production of neutralizing antibodies albeit generating peptide specific high titered antibodies (46, 47). Similarly, antisera raised against synthetic peptides corresponding to the N- and C-terminal heptad repeats in gp41 are nonneutralizing although they are capable of reacting with gp41 (for references see Golding et al., 2002; 48). Thus, epitope vaccines have so far been unsuccessful because they have failed to elicit neutralizing antibody production. Consistent with the capacity of neutralizing antibodies to control HIV infection, several studies have demonstrated that neutralizing antibodies directed against HIV-1 envelope glycoprotein could prevent infection in primates and accelerate clearance of cell-free virions from the blood (49, 50).
Thus, vaccines are currently being developed such as cellular vaccines, which stimulate T-cell immunity, antibody vaccines, peptide vaccines, naked DNA vaccines, multi-valent virus vaccines such as recombinant carnarypox vaccines and combination vaccines such as a DNA vaccine combined with a fowlpox vaccine. However, due to HIV's extraordinary mutability and evolution into multiple subtypes or clades worldwide, an HIV vaccine prepared from one HIV clade may not be effective against a different clade or even within a given clade.
To overcome the problems associated with different existing clades, novel vaccines have been developed and clinical trials are underway with a vaccine that incorporates HIV genetic material from clades A, B and C. However, this does not solve the problem of AIDS patients that have the remaining HIV clades D, E, F, G, H, I, J and O. It should be emphasized that autologous neutralizing antibodies can be isolated from patients eight to ten weeks after HIV infection. However after a period of one-year, the early virus population loses its sensitivity to neutralization as it becomes replaced by mutated strains that are resistant to neutralization (51). Thus an efficient immunogen for the generation of neutralizing antibody production should represent a conserved domain in HIV envelope glycoproteins, and which does not undergo selective pressure because of the essential function in the HIV infectious cycle.
The synthetic peptide inhibitor T20 (29) blocks cell fusion and HIV-1 entry into non-infected host cells by disrupting the conformational changes in gp41 during the HIV-induced fusion process. T-20, known as Fuzeon™, is currently being experimentally tested in HIV-infected individuals. However tolerance to long term administration of T-20 and its toxicity remain at this time, unknown. Moreover, there is a rapid emergence of resistant HIV-1 in patients receiving T-20 (52). Another disadvantage of T-20 is that the treatment protocol uses very high amount (100 mg) of this synthetic peptide administered daily in order to obtain a potent inhibition of HIV infection.
Due to the HIV-1 worldwide epidemic, the search for drug treatments and vaccines for HIV-1 has taken a prime stage over other medical diseases, including HIV-2. Treatments and vaccines for HIV-2 have not been fervently pursued as much as HIV-1 since HIV-2 infection occurs more slowly and there is lower viral load in HIV-2 infected persons.
Thus, there is a need in the art for vaccines and pharmaceutical compositions to treat and/or prevent all forms of immunodeficiency viruses such as HIV-1, HIV-2 and SIV for all clades.
There is also a need in this art to provide vaccines or pharmaceutical compositions to treat and/or prevent all forms of immunodeficiency viruses for all clades at lower costs, with fewer side effects and which have less drug resistance.
In one aspect, the present invention provides peptides and variants of such peptides capable of eliciting neutralizing antibodies that block HIV infection. In another aspect of the invention these peptides are antigens.
In another aspect, the present invention provides antigens and variants of such antigens that are capable of eliciting broadly neutralizing antibodies that block infection by various types and subtypes of HIV.
In still another aspect the present invention concerns the peptides or the antigens according to the invention associated covalently or non-covalently with peptides corresponding to the V3 loop and/or any HIV-1 or HIV-2 envelope protein or glycoprotein or/and a foreign antigen of interest such as HBs (surface antigen of the hepatitis B virus) as described in U.S. Pat. No. 5,314,808 or LSA3 antigen from Plasmodium falciparum as described in U.S. Pat. No. 6,191,270. In this regard, the present invention also includes mixtures of the peptides or antigens with other antigens known In the art.
In yet another aspect the present invention provides a vaccine for prevention of HIV-infection.
In still another aspect, the present invention provides a pharmaceutical composition as a therapeutic vaccine for treating HIV.
In yet another aspect, the present invention provides antibodies raised against the antigens and antigen variants. These antibodies are used in immunotherapy to prevent and/or treat HIV-infection. Neutralizing antibodies that are used in passive vaccines are also disclosed.
In still another aspect, the present invention provides natural neutralizing antibodies against the antigen and antigen variants. These natural antibodies are used in immunotherapy to prevent and/or treat HIV infection. Methods for the preparation of human monoclonal antibodies are also disclosed.
In yet another aspect, the present invention provides a method or use of the antibodies to detect HIV. Kits are also disclosed.
In still another aspect, the present invention provides a method to detect specific antibodies in HIV infected individuals. Kits are also disclosed.
In yet another aspect, the present invention provides a method of treating and/or preventing AIDS. Use of the antigens and antibodies to treat HIV is also disclosed.
In yet another aspect, the present invention provides a purified peptide comprising at least one of the sequences of SEQ ID Nos. 1 to 18 and their use to isolate anti-HIV molecules.
In yet another aspect, the present invention provides a complex of caveolin bound to the peptides of SEQ ID Nos. 1 to 9 and 11 to 18 to prevent HIV infection.
These and other aspects are achieved by the present invention as evidenced by the summary of the invention, description of the preferred embodiments and the claims.